螺旋藻-壳寡糖生物被膜抑制剂的制备、表征及活性评价

Preparation, characterization and activity evaluation of Spirulina-chitooligosaccharides capable of inhibiting biofilms

摘要:

病原菌形成的生物被膜严重威胁人类健康，显著增强了病原菌的耐药性，针对生物被膜的特效药物亟待研究。从虾、蟹壳等中提取得到的壳寡糖是一种天然碱性寡糖，具有良好的杀菌效果，但其对生物被膜的抑制作用仍有待提高。螺旋藻（Spirulina，SP）是一种表面带负电荷的微藻，其与壳寡糖形成的复合物可能发挥协同增效杀灭生物被膜深处病原菌的作用。针对提升壳寡糖的抑生物被膜作用，本研究首先通过浊度法筛选得到了杀菌效果显著的壳寡糖，并通过静电吸附作用将壳寡糖与螺旋藻结合，完成螺旋藻@壳寡糖（Spriulina@Chitooligosaccharides，SP@COS）复合物的制备。通过测定zeta电位、粒径和荧光标记等方法表征了壳寡糖和螺旋藻的结合情况，紫外-可见吸收光谱（ultraviolet-visible absorbance spectroscopy，UV-Vis）结果显示出螺旋藻对壳寡糖的包封率达90%，负载率达16%。制备的SP@COS对细菌、真菌生物被膜都有明显的增效抑制作用，且这种抑制效果主要是通过深入生物被膜内部、破坏细胞结构所实现。这些结果显示了螺旋藻-壳寡糖复合物具备作为生物被膜抑制剂的潜力，为提高壳寡糖的抑生物被膜作用、解决病原菌的危害提供了理论基础与新的思路。

Abstract:

The biofilms formed by pathogenic microorganisms seriously threaten human health and significantly enhance drug resistance, which urgently call for developing drugs specifically targeting on biofilms. Chitooligosaccharides extracted from shrimp and crab shells are natural alkaline oligosaccharides with excellent antibacterial effects. Nevertheless, their inhibition efficacy on biofilms still needs to be improved. Spirulina (SP) is a microalga with negatively charged surface, and its spiral structure facilitates colonization in the depth of the biofilm. Therefore, the complex of Spirulina and chitooligosaccharides may play a synergistic role in killing pathogens in the depth of biofilm. This research first screened chitooligosaccharides with significant bactericidal effects. Subsequently, Spirulina@Chitooligosaccharides (SP@COS complex was prepared by combining chitooligosaccharides with Spirulina through electrostatic adsorption. The binding of the complex was characterized by zeta potential, z-average size, and fluorescence labeling. Ultraviolet-visible spectroscopy (UV-Vis) showed the encapsulation efficiency and the drug loading efficiency reached up to 90% and 16%, respectively. The prepared SP@COS2 exhibited a profound synergistic inhibition effect on bacterial and fungal biofilms, which was mainly achieved by destroying the cell structure of the biofilm. These results demonstrate the potential of Spirulina-chitooligosaccharides complex as a biofilm inhibitor and provide a new idea for addressing the harm of pathogenic microorganisms.